Huntington's disease (HD) is an autosomal dominantly-inherited, neurodegenerative disorder caused by an expanded trinucleotide repeat (CAG) at the IT15 locus on chromosome 4. These repeats cause degeneration of striatal and cortical neurons in the brain resulting in a devastating cognitive, psychiatric, and motor disorder. RNA interference (RNAi) has emerged as a tool to decrease diseased gene expression. RNAi therapy involves the delivery of short hairpin RNAs (shRNAs) to affected cells which bind to and decrease the expression of target mRNA. It is a therapy extremely well-suited for diseases such as HD, which arise exclusively from a single, inherited gene mutation. To date, allele-specific silencing of mutant huntingtin (htt) remains unsuccessful because no RNAi-targetable polymorphism representing a large fraction of HD patients has been identified. Therefore, partial reduction of both the normal and the diseased allele should be tested as a therapy for HD. Preliminary studies in our lab demonstrate that short-term (4 months post-injection) adeno-associated viral delivery of three different shRNAs to the striata of CAG 140 mice results in an equal 50% partial reduction of htt. While all three hairpins were efficacious in partially reducing htt, two of the three hairpins induced striatal toxicity while the third did not. Toxicity was evidenced histologically as striatal atrophy and ventricular enlargement seen on Nissl and DARPP-32 stained tissue along with a robust increase in astrogliosis and a microglial response. The aims of this proposal are two- fold: 1) To address the question of why two of the three hairpins caused striatal toxicity even though they were designed using the same set of rules and were injected into the mouse striata at the same volume and titer. I hypothesize that, based on preliminary in vitro data, inappropriate strand biasing (loading both the sense and antisense strands of the hairpin) also occurs, in vivo, following viral delivery of the two toxic hairpins (sh2.4 and sh30.1), while only the correct antisense strand is loaded following viral delivery of the non-toxic hairpin (sh8.2); 2) To perform a long-term study using the non-toxic hairpin assessing the efficacy of partially reducing the expression of both htt alleles in the CAG 140 knock-in mouse as an extension of our preliminary data. Thorough behavioral, histological, molecular, and biochemical analysis will reveal whether a partial reduction of both alleles remains efficacious 10 months post-injection. Together, these two studies will address the molecular mechanisms behind differential cellular responses to viral injection of hairpins into the striatum and whether our non-toxic candidate hairpin is efficacious in the long-term, both of which are critical in developing RNAi as a therapy for HD, and more broadly, assessing RNAi in the mammalian brain. [unreadable] [unreadable] [unreadable]